利福平耐药基因突变位点的分析及M-ARMS-PCR检测方法的建立
发布时间：2019-07-05 15:26
【摘要】：背景研究 结核性脑膜炎由结核杆菌引起的中枢神经系统感染是最严重的肺外结核。虽然结核菌的耐药率在结核性脑膜炎患者中明显低于肺部感染患者，但是多耐药结核性脑膜炎的患者会遗留更严重的神经系统后遗症或者直接死亡，给社会和家庭造成了严重的负担。患者常会缺乏快速准确的诊断和药物敏感性检测，而延误治疗，最终导致严重后遗症、产生多耐药结核或使多耐药结核杆菌更进一步发展成为广泛性耐药，，使得结核控制产生了更严峻的挑战。WHO推荐结核杆菌感染患者需要早期接受正规，足量治疗，并且每个患者需接受药物敏感性检测性，并根据其耐药诊断，选择合理的药物治疗。规范的治疗在肺结核治疗中降低致残率和致死率并且提高预后和控制多耐药结核的发生率等方面都起到了明显的作用。但是目前结核性脑膜炎治疗中，由于没有明确的诊断和治疗指南，往往是根据肺结核的诊治方法及经验用药。且由于血脑屏障等因素，往往用药剂量和时程均大于肺结核，例如在结核性脑膜炎的经验治疗中利福平用药比肺结核治疗时，剂量大，时间长，没有规范治疗和用药则更有可能导致利福平耐药。但是由于结核性脑膜炎病例很难收集，脑脊液结核杆菌数量少，很难培养成功等现状，针对结核性脑膜炎患者的耐利福平结核分枝杆菌基因突变等机制研究及结核性脑膜炎耐药检测方法研究并不充分，更无法做到询证医学。所以多耐药结核性脑膜炎检测及机制还有需要更多的研究支持。 目的 鉴于肺结核与结核性脑膜炎治疗中利福平用药剂量及治疗时间上的差异，探究这两种患者中的利福平耐药的突变位点是否存在差异；建立一个快速，简单，高通量，低成本的检测方法可以检测结核分枝杆菌耐药突变基因，并用于结核性脑膜炎的诊断。 方法 通过测序的方法比较导致结核性脑膜炎的结核分枝杆菌利福平耐药基因突变机制与致肺结核结核分支杆菌耐药基因突变位点有无差别，并根据结核耐药数据库给予的结果，选取高度确信耐药位点，通过NCBI的GENE数据库获得rpoB基因（Rv0667），根据M-ARMS-PCR原理进行设计引物，设计五对引物分别为1037OF-1048OR；511IF-1478OR；516IF-1478OR；526IF-1478OR；531IF-1478OR，其中5个单独的上游引物，均与下游1478OR组成4对引物，分别形成半巢式PCR，产物大小分别：442bp、227bp、214bp、183bp、166bp。选取135例培养阳性并通过药敏检测的结核分支杆菌菌株，进行M-ARMS-PCR扩增，并应用QIAxcel仪器进行检测分析。同时针对这135例菌株的耐药决定区域进行DNA测序。应用药敏结果和测序结果对M-ARMS-PCR方法进行评价。 结果 本次研究通过测序的方法对比导致结核性脑膜炎利福平耐药的结核分枝杆菌与导致肺结核结核分枝杆菌利福平耐药rpoB基因突变的分布情况，102株利福平耐药的结核分枝杆菌的rpoB基因测序发现，63株导致肺结核的结核分枝杆菌的rpoB基因突变常见位点为：531（53.97%），526（20.63%），516（6.45%）。39株导致结核性脑脑膜炎的rpoB基因突变最常见位点为：531（61.54%），526（20.51%），533（7.69%）。建立了基于QIAxcel检测平台的利福平耐药基因rpoB检测的M-ARMS-PCR检测方法。这种方法能够单管5重选择性PCR扩增rpoB基因高度确信耐药四个密码子511、516、526、531的野生型基因，不扩增突变型。M-ARMS-PCR结果中，131例样品与DNA测序结果一致。用DNA测序方法评价M-ARMS-PCR方法的敏感度为94.2%，特异性为100%。药敏检测方法评价M-ARMS-PCR的方法的敏感度为86.57%，特异性为89.71%。 结论 通过本次试验了解了导致结核性脑膜炎的结核分支杆菌利福平耐药rpoB基因最常见的突变区域为531-533区域。其中531位密码子的Ser-Leu突变率占明显优势。 建立的基于QIAxcel检测平台的M-ARMS-PCR的检测方法检测利福平耐药基因突变，可以在6个小时能完成96个样品检测达到了预期实验目的。具有便捷，快速，准确，早期，廉价的优点，适宜应用于贫困，医疗水平相对落后的地区。
文内图片：
图片说明：具有代表性TBM样品测序峰图
[Abstract]:background research The central nervous system infection caused by tuberculosis of tuberculous meningitis is the most serious extrapulmonary junction. Nuclear. Although the resistance rate of the tubercle bacillus is significantly lower than that of the patients with pulmonary infection in the patients with tuberculous meningitis, the patients with multi-drug-resistant tuberculous meningitis left more serious neurological sequela or direct death, causing serious negative effects on the society and the family The patients often lack rapid and accurate diagnosis and drug sensitivity detection, and delay treatment, resulting in serious sequela, multi-drug-resistant tuberculosis or even further development of the multi-drug-resistant mycobacterium tuberculosis, so that the control of the tuberculosis is more severe. The WHO recommends that patients with M. tuberculosis infection require early acceptance of normal, adequate treatment, and each patient is subject to drug sensitivity detection and, based on its drug resistance diagnosis, select a reasonable drug treatment The treatment has played an important role in the treatment of pulmonary tuberculosis, the reduction of the disability rate and the mortality rate, the improvement of the prognosis and the control of the incidence of multi-drug-resistant tuberculosis, etc. In the treatment of tuberculous meningitis, there is no clear diagnosis and treatment guide, often based on the diagnosis and treatment of pulmonary tuberculosis and experience. As a result of the blood-brain barrier and other factors, the dosage and time history of the drug are more than that of the pulmonary tuberculosis, for example, when the rifampin is used in the treatment of the tuberculosis meningitis, the dosage is large, the time is long, the treatment and the medication are not standardized, and the rifampin resistance is more likely to be caused. However, because of the difficulty in collecting the cases of tuberculous meningitis, the number of tubercle bacillus in the cerebrospinal fluid is small, it is difficult to culture the present status, and the research on the mechanism of the detection of the resistance to the rifampin-resistant Mycobacterium tuberculosis in the patients with tuberculous meningitis and the method of the detection of the drug resistance of the tuberculous meningitis do not charge. No, I can't make an inquiry. Therefore, there are more studies on the detection and mechanism of multi-drug-resistant tuberculous meningitis. Hold on. Objective To investigate the difference in the dose of rifampin and the treatment time of rifampin in the treatment of tuberculosis and tuberculous meningitis. the detection method of the single, high-flux and low-cost can detect the mycobacterium tuberculosis drug-resistant mutant gene, synovitis The method of the invention compares the mycobacterium tuberculosis rifampin resistance gene mutation mechanism of the tuberculous meningitis and the mycobacterium tuberculosis mycobacterium tuberculosis drug resistance gene mutation site by the method of sequencing, and selects the drug resistance gene mutation site of the mycobacterium tuberculosis mycobacterium tuberculosis mycobacterium tuberculosis drug resistance gene mutation mechanism and the tuberculosis drug resistance database, The high-confidence resistance site was obtained. The rpoB gene was obtained through the GENE database of NCBI (Rv0667). The primers were designed according to the principle of M-ARMS-PCR. The five pairs of primers were 1037OF-1048OR, 511IF-1478OR, 55IF-1478OR, 526IF-1478OR, 531IF-1478OR, and 5 individual upstream primers were used to form 4 pairs of primers with the downstream 1478OR to form half-nested PCR and the product was large. Small:442 bp,227 bp,214 bp,18 The M-ARMS-PCR was amplified by M-ARMS-PCR and QIAxce was used. 1. Detection and analysis of the instrument. The drug resistance of these 135 strains was determined. DNA sequencing was carried out in the region. The results of drug sensitivity and sequencing showed that M-ARMS- PC Evaluation of R. Results The results of this study were compared to the distribution of rifampin-resistant Mycobacterium tuberculosis and rifampin-resistant rpoB gene. The most common site of rpoB gene mutation in tuberculous meningitis was 531 (53.97%),526 (20.63%) and 516 (6.45%), and the most common site of rpoB gene mutation in tuberculous meningitis was 531 (61.54%),526 (20.51 (%),533 (7.69%). The detection of rifampin-resistant gene rpoB based on the QIAxcel detection platform was established. -ARMS-PCR detection method. This method is capable of amplifying the rpoB gene by a single-tube 5-weight selective PCR with four codons 511,516,526,531, Wild-type gene, no mutant. M-ARMS-PCR result,131 Example samples were consistent with the DNA sequencing results. The sensitivity of the M-ARMS-PCR method was evaluated using a DNA sequencing method to be 9 The sensitivity of the method for evaluating M-ARMS-PCR was 86.5. 7% And the specificity is 89.71%. The common mutation area is the 531-533 area. The 531-bit password The detection method of M-ARMS-PCR based on QIAxcel detection platform detects rifampin resistance gene mutation and can be used for 6 hours. The method has the advantages of convenience, rapidness, accuracy, early and low cost,
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R529.3
文内图片：
图片说明：具有代表性TBM样品测序峰图
[Abstract]:background research The central nervous system infection caused by tuberculosis of tuberculous meningitis is the most serious extrapulmonary junction. Nuclear. Although the resistance rate of the tubercle bacillus is significantly lower than that of the patients with pulmonary infection in the patients with tuberculous meningitis, the patients with multi-drug-resistant tuberculous meningitis left more serious neurological sequela or direct death, causing serious negative effects on the society and the family The patients often lack rapid and accurate diagnosis and drug sensitivity detection, and delay treatment, resulting in serious sequela, multi-drug-resistant tuberculosis or even further development of the multi-drug-resistant mycobacterium tuberculosis, so that the control of the tuberculosis is more severe. The WHO recommends that patients with M. tuberculosis infection require early acceptance of normal, adequate treatment, and each patient is subject to drug sensitivity detection and, based on its drug resistance diagnosis, select a reasonable drug treatment The treatment has played an important role in the treatment of pulmonary tuberculosis, the reduction of the disability rate and the mortality rate, the improvement of the prognosis and the control of the incidence of multi-drug-resistant tuberculosis, etc. In the treatment of tuberculous meningitis, there is no clear diagnosis and treatment guide, often based on the diagnosis and treatment of pulmonary tuberculosis and experience. As a result of the blood-brain barrier and other factors, the dosage and time history of the drug are more than that of the pulmonary tuberculosis, for example, when the rifampin is used in the treatment of the tuberculosis meningitis, the dosage is large, the time is long, the treatment and the medication are not standardized, and the rifampin resistance is more likely to be caused. However, because of the difficulty in collecting the cases of tuberculous meningitis, the number of tubercle bacillus in the cerebrospinal fluid is small, it is difficult to culture the present status, and the research on the mechanism of the detection of the resistance to the rifampin-resistant Mycobacterium tuberculosis in the patients with tuberculous meningitis and the method of the detection of the drug resistance of the tuberculous meningitis do not charge. No, I can't make an inquiry. Therefore, there are more studies on the detection and mechanism of multi-drug-resistant tuberculous meningitis. Hold on. Objective To investigate the difference in the dose of rifampin and the treatment time of rifampin in the treatment of tuberculosis and tuberculous meningitis. the detection method of the single, high-flux and low-cost can detect the mycobacterium tuberculosis drug-resistant mutant gene, synovitis The method of the invention compares the mycobacterium tuberculosis rifampin resistance gene mutation mechanism of the tuberculous meningitis and the mycobacterium tuberculosis mycobacterium tuberculosis drug resistance gene mutation site by the method of sequencing, and selects the drug resistance gene mutation site of the mycobacterium tuberculosis mycobacterium tuberculosis mycobacterium tuberculosis drug resistance gene mutation mechanism and the tuberculosis drug resistance database, The high-confidence resistance site was obtained. The rpoB gene was obtained through the GENE database of NCBI (Rv0667). The primers were designed according to the principle of M-ARMS-PCR. The five pairs of primers were 1037OF-1048OR, 511IF-1478OR, 55IF-1478OR, 526IF-1478OR, 531IF-1478OR, and 5 individual upstream primers were used to form 4 pairs of primers with the downstream 1478OR to form half-nested PCR and the product was large. Small:442 bp,227 bp,214 bp,18 The M-ARMS-PCR was amplified by M-ARMS-PCR and QIAxce was used. 1. Detection and analysis of the instrument. The drug resistance of these 135 strains was determined. DNA sequencing was carried out in the region. The results of drug sensitivity and sequencing showed that M-ARMS- PC Evaluation of R. Results The results of this study were compared to the distribution of rifampin-resistant Mycobacterium tuberculosis and rifampin-resistant rpoB gene. The most common site of rpoB gene mutation in tuberculous meningitis was 531 (53.97%),526 (20.63%) and 516 (6.45%), and the most common site of rpoB gene mutation in tuberculous meningitis was 531 (61.54%),526 (20.51 (%),533 (7.69%). The detection of rifampin-resistant gene rpoB based on the QIAxcel detection platform was established. -ARMS-PCR detection method. This method is capable of amplifying the rpoB gene by a single-tube 5-weight selective PCR with four codons 511,516,526,531, Wild-type gene, no mutant. M-ARMS-PCR result,131 Example samples were consistent with the DNA sequencing results. The sensitivity of the M-ARMS-PCR method was evaluated using a DNA sequencing method to be 9 The sensitivity of the method for evaluating M-ARMS-PCR was 86.5. 7% And the specificity is 89.71%. The common mutation area is the 531-533 area. The 531-bit password The detection method of M-ARMS-PCR based on QIAxcel detection platform detects rifampin resistance gene mutation and can be used for 6 hours. The method has the advantages of convenience, rapidness, accuracy, early and low cost,
【学位授予单位】：第四军医大学
【学位级别】：硕士
【学位授予年份】：2013
【分类号】：R529.3
【参考文献】
相关期刊论文 前2条
1 刘颖;张晨;王淼;吴希阳;马学军;;基于四引物扩增受阻突变体系PCR的多重乳腺癌相关SNP位点检测方法的建立[J];中国科学:生命科学;2012年02期
2 黄海荣,金奇,马s
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